Long-Read Sequence Confirmed a Large Deletion Including MYH6 and MYH7 in an Infant of Atrial Septal Defect and Atrial Arrhythmias.

概要

MYH6, encoding α-cardiac myosin heavy chain, is one of the responsible genes for atrial septal defect (ASD) 1. Although single nucleotide variants (SNVs) in MYH6 have been reported as the cause of ASD, there are few literatures describing DNA structural variants (SVs) which are defined as more than 50 bp regions of DNA showing deletions, insertions, duplications, inversions or translocations. Short-read sequencing (SRS) can detect SNVs with high accuracy but SVs with lower sensitivity2, therefore, it might overlook SVs. The read length feasible by long-read sequencing (LRS) is tens to thousands of kilobases, and LRS can detect the breakpoints of complex SVs 3 .In this study, we confirmed a large deletion extending from MYH6 to MYH7 including the breakpoints in a family with inherited ASD, using both SRS and LRS.

The proband was a five-month-old boy with ASD detected by echocardiography (Fig.A), atrial flutter (AFL) and tachycardia. He received catheter ablation therapy for AFL at two. Although his voltage map of both atriums showed no myocardial damages, the right atrium was remarkably enlarged (Fig. B). His mother had ASD which was closed spontaneously. There were no findings indicating cardiomyopathy in the proband and his mother.

After obtaining the consent from his parents for the genetic analysis approved by our institutional review board, we performed targeted SRS for 58 genes related to inherited cardiac arrhythmias. The proband’s SRS did not identify any responsible SNVs. To detect deletions and duplications, we compared the read depth between the proband and control sample (pair analysis) using SureCall software (v. 2.1.2.11) (Agilent, Santa, CA, USA). The log ratios from exon 26 to exon 3 in MYH6 were lower than that of the normal range, which meant the deletion of these exons (Fig.C). Because a deletion between MYH6 intron 25 and MYH7 intron 26 was reported as esv2748480 and nsv4228245 on dbVar (https://www.ncbi.nlm.nih.gov/dbvar/), we performed long-range PCR from MYH6 intron 26 to MYH7 exon 26 and found an abnormal PCR product with the size of 1.5K bases in the proband and his mother. The sequence from chr14:23390033 to 23390448 including MYH6 exon 26 is identical to chr14:23419821 to 23420236 including MYH7 exon 27 (Fig. D, yellow box indicating the homologous part). Although we designed primers at the specific sequence in each gene, it is difficult to read the sequence over the homologous part at once and we could not confirm the deletion.

To resolve this problem, we performed whole-genome LRS with the Nanopore sequencer GridION X5 (Oxford Nanopore Technologies, Oxford, UK). The long reads were aligned to the human reference genome sequence (build hg38) by NGMLR (v.0.2.7) 4. Sniffles (v.1.0.11) 4 , calling any types of SVs, detected a deletion encompassing from chr14: 23390037 to 23419824 (hg38) (Fig. D and Fig. E). We reconfirmed the result of Sanger sequencing to determine the precise positions connecting the homologous part and the specific part of each gene. Based on 3’ rule, we concluded that a range of the deletion was from chr14:23390449 to 23420236, which produced the same DNA rearrangement as the deletion detected by Sniffles (Fig. D).

This is the first report showing a large deletion between MYH6 and MYH7 in a family with ASD. Several missense and truncating variants in MYH6 have been reported as responsible for ASD. In animal models, morpholino knock-down of expression of the chick MYH6 homolog eliminates the formation of the atrial septum without overtly affecting atrial chamber formation. The variants in TBX5 and GATA4, which are associated with expression of MYH6, have been reported as causative for ASD. These literatures suggested MYH6 downregulation would be associated with ASD. In our patient, a new hybrid protein consisting of MYH6 and MYH7 might be produced because this deletion is in-frame, and hybrid protein was possibly under control of the MYH7 promoter unaffected by the deletion. It may lead the expressions of reduced normal MYH6 and of unbalanced MYH6/MYH7 in the atria. Unfortunately, the cardiac tissue of the proband was not available and we could not confirm our hypothesis.

Nsv4228245 and esv2748480 are almost same as our deletion. Although esv2748480 was found in 4 participants in 100 Malays, the cohort was small, and 4 might be relatives. In gnomAD, nsv4228245 was found in only 2 of 20310 alleles (MAF= 9.8e-005) 5 and is enough rare to be considered as a clinically relevant variant. Moreover, most patients with ASD are asymptomatic, and ASD sometimes close spontaneously, therefore the participants with these SVs might have ASD latently.

In this study, we identified a large deletion using both SRS and LRS methods. As in our case, SVs including high homology sequence would be one of the suitable targets of Nanopore sequencing. The combination of SRS and LRS is useful to confirm the detail of SVs in patients with suspected inherited diseases but carrying no causative SNVs.

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